Intraoperative use of enriched collagen and elastin matrices with freshly isolated adipose-derived stem/stromal cells: a potential clinical approach for soft tissue reconstruction

Abstract

Background: Adipose tissue contains a large number of multipotent cells, which are essential for stem cell-based therapies. The combination of this therapy with suitable commercial clinically used matrices, such as collagen and elastin matrices (i.e. dermal matrices), is a promising approach for soft tissue reconstruction. We previously demonstrated that the liposuction method affects the adherence behaviour of freshly isolated adipose-derived stem/stromal cells (ASCs) on collagen and elastin matrices. However, it remains unclear whether freshly isolated and uncultured ASCs could be directly transferred to matrices during a single transplantation operation without additional cell culture steps.

Methods: After each fat harvesting procedure, ASCs were isolated and directly seeded onto collagen and elastin matrices. Different time intervals (i.e. 1, 3 and 24 h) were investigated to determine the time interval needed for cellular attachment to the collagen and elastin matrices. Resazurin-based vitality assays were performed after seeding the cells onto the collagen and elastin matrices. In addition, the adhesion and migration of ASCs on the collagen and elastin matrices were visualised using histology and two-photon microscopy.

Results: A time-dependent increase in the number of viable ASCs attached to the collagen and elastin matrices was observed. This finding was supported by mitochondrial activity and histology results. Importantly, the ASCs attached and adhered to the collagen and elastin matrices after only 1 h of ex vivo enrichment. This finding was also supported by two-photon microscopy, which revealed the presence and attachment of viable cells on the upper layer of the construct.

Conclusion: Freshly isolated uncultured ASCs can be safely seeded onto collagen and elastin matrices for ex vivo cellular enrichment of these constructs after liposuction. Although we observed a significant number of seeded cells on the matrices after a 3-h enrichment time, we also observed an adequate number of isolated cells after a 1-h enrichment time. However, this approach must be optimised for clinical use. Thus, in vivo studies and clinical trials are needed to investigate the feasibility of this approach.

Figure 1

An illustration of the described approach. After isolation of cells from adipose tissue secondary to liposuction, the obtained adipose-derived stem/stromal cells were directly transferred to the collagen and elastin matrix without additional cell culture steps. The cells were incubated on top of the material for different clinically relevant time periods (i.e., 1 and 3 h) for cellular matrix enrichment. For comparison, the cells were incubated with the matrices for 24 h.

Figure 2

Adherence of viable adipose-derived cells onto collagen and elastin matrices. The box-and-whisker plots display the fluorescence signals obtained from adhered and viable adipose-derived cells at different time points (i.e., 1, 3, and 24 h) after seeding on collagen and elastin matrices, as indicated by the alamarBlue resazurin-based assay. The bottom and top of the box represent the first and third quartiles, the band inside the box represents the median, and the whiskers represent the minimum and the maximum of the data. All data values are presented in the results section. Details related to the statistical analysis are presented in the Methods section. n = 10, *p < 0.05, arbitrary unit; AU.

Figure 3

Histology of enriched collagen and elastin matrices. This figure shows representative histology images of the collagen and elastin matrices, which were incubated with freshly isolated adipose-derived stem/stromal cells for the indicated time periods (scale bar is 200 μm).

Figure 4

Two-photon microscopy of the enriched collagen and elastin matrices. This figure shows a representative two-photon microscopy image of a collagen and elastin matrix, which was incubated with freshly isolated adipose-derived stem/stromal cells for only 1 h. The green colour indicates the cytoplasm of viable cells, which was stained using fluorescein diacetate (FDA), the blue colour indicates the cell nuclei, which were stained using Hoechst 33342, and the grey colour indicates the collagen and elastin structure (scale bar is 100 μm).